The present invention, in some embodiments thereof, relates to novel anti-microbial agents and more particularly, but not exclusively, to novel agents that are capable of preventing biofilm formation and/or reducing biofilm mass, and to uses thereof in various applications.
Many bacteria are planktonic, namely they move freely around in water and other liquid media, however, many pathogenic and harmful microorganisms are or become sessile, namely attached to a surface where they form biofilms. Biofilms are a multicellular high density ecological environment of mostly bacteria and/or fungi and their secretions, but it is not considered a multicellular organism per se. Once a microorganism attach to a surface it undergoes a series of changes, the most obvious of which is the excretion of a slimy material consisting mostly of extra-cellular polysaccharides (EPS). EPS, soluble microbiological products (SMP), dispersed bacterial cells, and a well characterized natural organic matter (NOM) have all been identified as part of a “conditioning layer” that may help retain other fouling materials, as well as directly cause membrane biofouling.
A dramatic phenotypic change occurs as the bacteria turn or switch from a planktonic to a biofilm state and attach to a surface; a whole different suite of genes is activated, making sessile bacteria significantly different to planktonic bacteria suspended in the water. Biofilm bacteria have been found to remain viable at MICs (minimal inhibitory concentration) up to 1,000 times higher than those of their planktonic counterparts. Biofilms have been shown to alter the local environment to enhance their survival, changing such properties as pH and the dissolved oxygen concentration. These changes can reduce the effectiveness of some treatments. For example, biofilms are known to vary the local pH, and some oral biofilms have regions of pH less than 4.9.
In addition to their complex, heterogeneous composition, biofilms are also dynamic hydrogels which capriciously move, detach and reform on a wide variety of environmental or engineered surfaces. Thus, when water-borne bacteria congregate in sufficient numbers they may form a film on the surface of pipes, tanks, and indeed any piece of equipment, and biofouling usually results. Biofouling can be defined as the unspecific adsorption of biological material onto surfaces upon their immersion in a fluid. EPS secreted by bacteria and other colonizing microorganisms envelope and anchor them to the substrate thereby altering the local surface chemistry which can stimulate further growth such as the recruitment and settlement of microorganisms. Biofouling via biofilm formation causes the deterioration in the microbiological quality of water by inducing biocorrosion termed microbiologically influenced corrosion and biofouling of piping, membranes, containers and reservoirs.
Biofilms also interrupt the flow of ions and water to and from the substrate surface by acting as a diffusion barrier. The reduction of localized oxygen by cathodic reactions within the electrolyte can accelerate the corrosion of a metallic substrate by creating a differential aeration concentration cell. The corrosion and weathering caused by biofilm can lead to considerable damage to heat exchangers, unexpected corrosion of stainless steel, and premature destruction of membranes, and many other technological, industrial and homestead aliments.
Both bacteria and fungi share the same habitat in the oral cavity, although they belong to different kingdoms in the evolution hierarchy. They both harbor mixed biofilms which cover oral tissues. Streptococcus mutans is a cardinal member of the oral biofilm associated with dental caries while Candida albicans is associated with oral candidiasis. Bacteria can communicate therebetween by what is known as quorum sensing (QS) which is effected by secreting small molecules termed auto inducers (AI's) into their environment. This phenomenon affects many physiological and metabolic pathways of bacteria, including the formation of biofilms and antibacterial resistance. Recently, eukaryotic cells such as fungi have also been shown to communicate with each other by producing AIs, however, the AIs of bacteria and fungi differ chemically. Quorum sensing takes place especially in biofilms were the microbes are at close proximity to one another.
Inter species QS may affect microbes' physiology and virulence properties resulting in enhanced virulent properties of biofilms. Small peptides, AI-2 (furanosyl borate diester), AI-1 (N-acylhomoserine lactones) and C-AI (Cholera AI) have been shown to act as signal molecules in QS in many types of bacteria, including oral bacteria.
The study of bacterial QS has suggested several ideal targets for manipulation of QS, mainly the AI-2 signal molecule or its sensor-2, due to the wide distribution of the AI-2 cascade in many types of bacteria.
Similarly, it has been reported recently that also fungi communicate between themselves with signal molecules, and production of farnesol by C. albicans at high cell densities is the first QS system which has been discovered in eukaryotes. Farnesol has been identified as QS agent that blocks the morphological transition from yeast to the filament form and affects biofilm formation in C. albicans. The mechanism by which farnesol is sensed by C. albicans is not yet known. Farnesoic acid and tyrosol were also shown to posses AI properties in C. albicans. 
Eukaryotes seem to have evolved efficient mechanisms to manipulate bacterial QS and thereby protect themselves from pathogenic bacterial attack and competition [Hogan, D. A., 2006, Eukaryot Cell, 5, 613-9]. Thus, by producing quorum sensing inhibitors (QSIs), the eukaryotic host may be simultaneously conversing with a variety of different bacterial strains that it encounters in its natural habitat, potentially encouraging the beneficial ones and antagonizing the harmful strains. Parallel to this, certain bacteria have evolved mechanisms to fine-tune gene regulation of eukaryotic with their QS signals [Hogan D. A. et al., 2004, Mol. Microbiol., 54, 1212-23]. This eukaryote-bacterial cross talk could be exploited to model manipulative techniques that interfere with bacterial QS.
Hence, it has been established that bacteria and fungi immobilized in the form of biofilms are inherently more robust and resistant to antibiotics and antifungal agents than the planktonic forms. Those mixed biofilms constitute a basis of numerous infections and diseases and are responsible in part to the growing emergence of resistance to antimicrobial agents. Thus, small compounds that can interfere with QS can offer a new approach to the development of novel antimicrobial and anti-biofilm agents. It is now evident that influencing QS in microbes harboring biofilms bears great potential as a novel non-antimicrobial, alternative means of affecting pathogenic microbes.
Cell harvesting and tissue dissociation is widely used in science and medicine. Harvesting single cells, for study, analyses, identification, pathology, processing or subculture propagation, either of unicellular or multicellular organisms, oftentimes requires dissociation or detachment of single cells from a biofilm or a tissue.
While cell harvesting is a routine procedure in handling and propagating tissue culture cell lines, tissue disaggregation is used for the establishment of primary cultures, and for the release of cell from a whole tissue for medical treatments. Primary cultures are a widely used tool in molecular biology, toxicology, and biotechnology. An example of cells release for medical use is the isolation of fibroblasts from skin biopsy for future skin transplantations. The tissue dispersion into a cell suspension is usually achieved by a combined mechanical and enzymatic procedure. The enzyme should be able to degrade the extra-cellular matrix components, such as collagen. The enzymatic procedure is frequently performed in combination with ion chelator such as EDTA.
The most commonly used enzyme for tissue disaggregation and cell harvesting is porcine- or bovine-derived trypsin. It was formerly thought that trypsin preparations simply hydrolyzed a proteinaceous adhesive bonding substance responsible for the tenacious attachment of cells to their substratum with the resultant detachment of the cells from the culture vessel. It is now assumed that the mechanism of action of trypsin in cell harvesting is more complex. Trypsin is most frequently used since it is effective for many tissues, and demonstrated to be well tolerated by many cell types, however, the exposure of cells to active enzymes should be minimized to preserve maximum viability, and hence trypsin inhibitors are used in most procedures.
Other methods for cells harvesting and tissue dissociation involve the use of plant or bacterial enzymes, such as Clostridium histolyticum collagenases, plant cardosins, and the commercial microbially-produced product GIBCO™ Protease™.
The presence of a thiazolidine ring in penicillins and related derivatives was the first recognition of its occurrence in nature, and since then many thiazolidine derivatives have shown antifungal or antibacterial activity [Heerding D. A. et al., 2003, Bioorg Med Chem Lett., 13, 3771-3].
The chemistry of thiazolidinediones (TZDs) has been extensively reviewed [Lesyk R. B. and Zimenkovsky B. S., 2004, Current Organic Chemistry, 8, 1547-77]. Furthermore, the interest in 2,4-thiazolidinedione derivatives has been heightened markedly during the last years due to their potential use as a new class of antidiabetic (insulin-sensitizing) agents (troglitazone, pioglitazone and darglitazone), that are currently used for the treatment of type-II Diabetes Mellitus.
(Z)-3-(octan-2-ylidene)-5-thioxopyrrolidin-2-one and (Z)-1-(hydroxymethyl)-3-(octan-2-ylidene)-5-thioxopyrrolidin-2-one, two thioxo derivatives of the short-alkyl TZDs (Z)-3-(octan-2-ylidene)pyrrolidine-2,5-dione and (Z)-1-(hydroxymethyl)-3-(octan-2-ylidene)-5-thioxopyrrolidin-2-on, respectively, have been reported by Ginak, A. I. et al. [Russian Journal of General Chemistry (Translation of Zhurnal Obshchei Khimii), 73(10), 1663-1664; 2003].
Pyrrolidinediones have been investigated for antibacterial activity and have shown a unique mechanism of action. The chemistry and activity of pyrrole-2,5-diones (PYDs) has been reviewed by, for example, Pohlmann, J. et al., Bioorganic and Medicinal Chem. Let., 15 (2005), pp. 1189-1192; Freiberg, et al., Antimicrobial Agents And Chemotherapy, 2006, pp. 2707-2712; Ozinskas A. J. et al., J. Org. Chem., 1986, 51, 26, pp. 5047-5050; and Mizufune, H. et al., Tetrahedron, 62 (2006), pp. 8539-8549.
WO 95/35296 discloses condensed imidazole compounds, their production and use as adhesion molecule expression inhibitors, and in passing mentions the intermediate compound (Z)-3-butylidenepyrrolidine-2,5-dione as a reactant or a reagent, which is a short-alkyl PYD derivative.
Barrett, Anthony G. M. et al. [J. Org. Chem., 1984, 49, 19, pp 3673-4 and J. Org. Chem., 1986, 51, 4, pp 495-503] report the synthesis of showdomycin and epi-showdomycin by cyclization of (E)-3-(2,3,4,5-tetrahydroxypentylidene)pyrrolidine-2,5-dione, and in passing mentions (E)-3-(4 hydroxybutylidene)pyrrolidine-2,5-dione, (Z)-3-(5-hydroxyhexylidene)pyrrolidine-2,5-dione and (E)-3-(4-hydroxypentylidene)pyrrolidine-2,5-dione, all of which are short-alkyl PYD derivatives.